Post-translational modifications of protein are hot theme in proteomic study.
Protein phosphorylation is a common and very important post translation of modification of protein. The phosphorylation and dephosphorylation of protein regulate nearly every aspects of cellular life including cell proliferation, development and differentiation, nerve activity, muscle shrinkage, metabolism and tumor progression etc. Protein phosphorylation and dephosphorylation also is well known as primary signal transduction pathway.
Traditional analysis methods of protein phosphorylation such as radio isotope labeling, Edman degradation and thin layer chromatography are often time-consuming. These methods not only need complicated manipulation, skilled-operator and large amount of proteins, but also have potential radioactivity danger. Nowadays mass spectrometry has been developed as one of the most powerful tool for analysis of protein phosphorylation. Identification of phosphoprotein by mass spectrometry is still a huge challenge. This is because, firstly, the phosphoprotein is often low abundance in cellular proteins; secondly, the negative charge of phosphopeptide is very difficult to ionization during mass spectrometric detection; and thirdly, the signals of phosphopeptides are often greatly suppressed by lots of non-phosphopeptides in the tryptic digest. Therefore, it is very difficult to directly apply mass spectrometer to analyze phosphopeptides in complex tryptic digest. So isolation and enrichment of phosphopeptides from complex peptides prior to mass spectrometry analysis is required.
By far, immobilized metal affinity chromatography (IMAC) is widely applied to enrich and isolate phosphopeptide. With IMAC technology, the chelating groups such as the iminodiacetic acids are often covalent linked to chromatography media, then metal ions (usually Fe3+ or Ga3+) are immobilized on the chromatography media because of strong chelation. The isolation of phosphopeptides with IMAC is achieved by the strong interaction between the phosphate groups on the phosphopeptides and the immobilized metal ions. However, an obvious disadvantage of this approach is its low specificity as some of acidic non-phosphopeptides are also retained. Those non-phosphopeptides may seriously suppress the detection of phosphopeptide. To improve specificity of phosphopeptide isolation, TiO2 and ZrO2 microparticle packed micro column, Al(OH)3 and Fe3O4/TiO2 core/shell nanoparticles were recently used to enrich and isolate phosphopeptides.
Reference 1 (Katz, H. E. et al., “Quaterthiophenediphosphonic (QDP): A Rigid, Electron-Rich Building Block for Zirconium-Based Multilayers”, Chemistry of Materials, P 699-703 (1991)) disclosed the method for the preparation of single or multi-layers of zirconium phosphonate surface by making use of the interaction between zirconium ion and phosphonate group. Also, Reference 2 (Guillaume Nonglation et al., “New approach to Oligonucleotide Microarrays Using Zirconium Phosphonate-Modified Surfaces”, Journal of American Chemical Society, P 1497-1502 (2004)) reported a method to prepare DNA microarray by taking advantage of the strong interaction between zirconium phosphonate and phosphonate group. Unfortunately, there is no literature describing or revealing the phosphopeptide isolation and enrichment method based on the strong interaction between zirconium phosphonate and phosphate groups.